Gamma-secretase inhibition reduce apoc3 levels and plasma triglycerides

ABSTRACT

A method of reducing a subject&#39;s plasma triglyceride level, comprising administering to a subject in need thereof a gamma-secretase inhibitor in an amount effective to reduce the subject&#39;s plasma triglyceride level.

This application claims the priority of U.S. Provisional Application No. 62/032,324, filed Aug. 1, 2014, and U.S. Provisional Application No. 61/936,279, filed Feb. 5, 2014 the contents of which are hereby incorporated by reference.

This invention was made with government support under grant number DK093604 awarded by the National Institutes of Health. The U.S. government has certain rights in the invention.

This application incorporates-by-reference nucleotide and/or amino acid sequences which are present in the file named “150204_0575_86108-A-PCT_SequenceListing_MW.txt,” which is 161 kilobytes in size, and which was created Feb. 4, 2015 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the text file filed Feb. 4, 2015 as part of this application.

All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual publication or reference were specifically and individually indicated to be incorporated by reference. Publications and references cited herein are not admitted to be prior art.

BACKGROUND OF THE INVENTION

Obesity has reached epidemic status in the United States. The Centers for Disease Control has stated that more than ⅓ of American adults are obese, and estimated the medical costs attributable to obesity at $147 billion in 2008, a number that is likely to be significantly higher today and in the future. One of the core components of obesity-induced metabolic syndrome is excess plasma triglycerides. Elevated plasma triglyceride level, or hypertriglyceridemia, is an independent risk factor for coronary heart disease, above and beyond other obesity-related complications (e.g., high LDL cholesterol, low HDL cholesterol and Type 2 Diabetes). Many patients are unable to get to plasma triglyceride targets with currently available therapies.

Thus, new therapies for reducing plasma triglycerides are needed.

SUMMARY OF THE INVENTION

The invention provides a method of reducing a subject's plasma triglyceride level, comprising administering to a subject in need thereof a gamma-secretase inhibitor in an amount effective to reduce the subject's plasma triglyceride level.

The invention also provides a method of treating a subject afflicted with hypertriglyceridemia, comprising administering to the subject a gamma-secretase inhibitor in an amount effective to treat the subject.

The invention also provides a method of reducing a subject's plasma glucose level, comprising administering to a subject in need thereof a gamma-secretase inhibitor in an amount effective to reduce the subject's glucose level.

The invention also provides a method of reducing a subject's ApoC3 level, comprising administering to a subject in need thereof a gamma-secretase inhibitor in an amount effective to reduce the subject's ApoC3 level.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Bifurcation model of hepatic insulin signaling illustrating how reduction in Notch signaling with γ-secretase inhibitor (GSI) treatment blocks mTorc1 activation, and tumorigenesis.

FIG. 2: Gamma-secretase inhibitors (GSIs) are pharmacologic inhibitors of Notch signaling.

FIG. 3: Plasma glucose levels of GSI and vehicle treated mice. *p<0.05**p<0.01***p<0.001 vs. vehicle.

FIG. 4: Serum triglyceride levels in mice treated with GSI or vehicle only. *p<0.05**p<0.01***p<0.001 vs. vehicle.

FIG. 5: VLDL, LDL, and HDL fraction serum triglyceride levels of mice administered GSI or vehicle only. *p<0.05**p<0.01***p<0.001 vs. vehicle.

FIG. 6: Lower plasma TG levels with low dose DBZ not associated with apparent GI toxicity. *p<0.05**p<0.01***p<0.001 vs. vehicle.

FIG. 7: Time course of plasma triglyceride levels of mice administered GSI or vehicle only.

FIG. 8: Time course of plasma triglyceride excursion following lipid gavage in mice administered GSI or vehicle only. *p<0.05**p<0.01***p<0.001 vs. vehicle.

FIG. 9: Glucose tolerance test (GTT) and pyruvate tolerance test (PTT) plots for control and L-Ncst mice. *p<0.05**p<0.01***p<0.001 vs. Cre-.

FIG. 10: Serum triglyceride levels for fasted and refed chow-fed L-Ncst mice.

FIG. 11: Serum lipid levels for control and L-Ncst mice. *p<0.05**p<0.01***p<0.001 vs. Cre-.

FIG. 12: Triglyceride levels by plasma fraction for control and L-Ncst mice. *p<0.05**p<0.01***p<0.001 vs. Cre-.

FIG. 13: Plasma triglyceride levels of fasted and refed control and L-Ncst mice. *p<0.05**p<0.01***p<0.001 vs. Cre-.

FIG. 14: Serum triglyceride levels in HFD-fed control and L-Ncst mice.

FIG. 15: Time course of serum triglyceride levels in control and L-Ncst mice. *p<0.05**p<0.01***p<0.001 vs. Cre-.

FIG. 16: Relative gene expression levels of fasted and refed control and L-Ncst mice.

FIG. 17: Western blot for ApoC3 serum levels in HFD-fed control and L-Ncst mice.

FIG. 18: Western blot for ApoC3 levels in HDL and VLDL serum fractions in HFD-fed control and L-Ncst mice.

FIG. 19: Western blot for hepatic ApoC3 levels in control and L-Ncst mice.

FIG. 20: Western blot for hepatic ApoC3 levels in L-Ncst mice.

FIG. 21: mRNA expression of Nicastrin and ApoC3 in fasted and refed control and L-Ncst mice.

FIG. 22: Correlation between ApoC3 (hepatic Apoc3 mRNA, hepatic ApoC3 protein, and serum ApoC3) with Plasma triglyceride.

FIG. 23: Serum triglyceride levels in HFD-fed control and L-Ncst mice following liver ApoC3 knockdown by adeno-delivered shRNA. *p<0.05**p<0.01***p<0.001 vs. Cre-.

FIG. 24: Adenoviral transduction of L-Ncst mice with ApoC3 increases plasma TG to levels comparable to Cre- control mice. p<0.05**p<0.01***p<0.001 vs. Cre-Ad-GFP mice.

FIG. 25: shRNA-mediated knockdown of Nicastrin (sequence of shRNA: CTCCTTCCACAATCGGTATTA) in rat hepatocytes reduces ApoC3 secretion.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of reducing a subject's plasma triglyceride level, comprising administering to a subject in need thereof a gamma-secretase inhibitor in an amount effective to reduce the subject's plasma triglyceride level.

The invention also provides a method of treating a subject afflicted with hypertriglyceridemia, comprising administering to the subject a gamma-secretase inhibitor in an amount effective to treat the subject.

In some embodiments, the administration reduces the triglyceride level in the subject's very low-density lipoprotein (VLDL) plasma fraction.

In some embodiments, the administration reduces the subject's plasma triglyceride level and apolipoprotein C3 (ApoC3) level.

In some embodiments, the administration reduces the subject's plasma triglyceride level and serum apolipoprotein C3 (ApoC3) level.

In some embodiments, the administration reduces the subject's serum triglyceride level.

In some embodiments, the administration reduces the triglyceride level in the subject's very low-density lipoprotein (VLDL) serum fraction.

In some embodiments, the administration reduces the subject's serum triglyceride level and apolipoprotein C3 (ApoC3) level.

In some embodiments, the administration reduces the subject's serum triglyceride level and serum apolipoprotein C3 (ApoC3) level.

The invention also provides a method of reducing a subject's ApoC3 level, comprising administering to a subject in need thereof a gamma-secretase inhibitor in an amount effective to reduce the subject's ApoC3 level.

In some embodiments, the administration reduces the subject's hepatic ApoC3 level.

In some embodiments, the administration reduces the subject's plasma ApoC3 level.

In some embodiments, the administration reduces subject's serum ApoC3 level.

In some embodiments, the administration reduces the ApoC3 level in the subject's HDL or VLDL serum fraction.

In some embodiments, the administration reduces the ApoC3 level in the subject's VLDL serum fraction.

In some embodiments, the administration reduces the subject's serum ApoC3 level and serum triglyceride level.

The invention also provides a method of reducing a subject's plasma glucose level, comprising administering to a subject in need thereof a gamma-secretase inhibitor in an amount effective to reduce the subject's glucose level.

In some embodiments, the administration the gamma-secretase inhibitor inhibits whole-body gamma-secretase.

In some embodiments, the administration of the gamma-secretase inhibitor inhibits liver gamma-secretase without significantly inhibiting gamma-secretase elsewhere in the subject.

In an embodiment, administration of the gamma-secretase inhibitor inhibits liver gamma-secretase without significantly inhibiting whole-body gamma-secretase.

In some embodiments, the administration of the gamma-secretase inhibitor targets the gamma-secretase inhibitor to the liver.

In some embodiments, the administration of the gamma-secretase inhibitor targets the gamma-secretase inhibitor to hepatocytes.

In some embodiments, the gamma-secretase inhibitor is (i) coupled to a ligand molecule targeted to a receptor on a hepatic cell, or (ii) administered by a bio-nanocapsule.

In some embodiments, the gastrointestinal Notch inhibition is substantially uninhibited.

In some embodiments, the gamma-secretase inhibitor is a small molecule inhibitor, an antisense oligonucleotide, or an adenoviral vector.

In some embodiments, the gamma-secretase inhibitor is a small molecule inhibitor, an oligonucleotide or an adenoviral vector.

In some embodiments, the gamma-secretase inhibitor is an oligonucleotide.

In some embodiments, the oligonucleotide is an antisense oligonucleotide, an RNA-interference inducing compound, or a ribozyme.

In some embodiments, the oligonucleotide is targeted to hepatocytes.

In some embodiments, the oligonucleotide comprises 1, 2, 3, 4, or 5 or more stretches of nucleotides in a sequence that is complementary to nicastrin-encoding mRNA, presenilin 1-encoding mRNA and presenilin 2-encoding mRNA, or APH1A-encoding mRNA and APH1B-encoding mRNA, wherein each stretch of complementary continguous nucleotides is at least at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides in length.

In some embodiments, the oligonucleotide comprises 1, 2, 3, 4, or 5 or more stretches of nucleotides in a sequence that is complementary to nicastrin-encoding mRNA, presenilin 1-encoding mRNA, presenilin 2-encoding mRNA, APH1A-encoding mRNA, or APH1B-encoding mRNA, wherein each stretch of complementary continguous nucleotides is at least at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides in length.

In some embodiments, the oligonucleotide is modified to increase its stability in vivo.

In some embodiment the small molecule inhibitor is 804929097, PF-3084014, BMS-708163, LY450139, or MK-0752.

In some embodiments, the gamma-secretase inhibitor is a small molecule inhibitor.

In some embodiments, the small molecule inhibitor is 2,2-dimethyl-N-((S)-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)-N′-(2,2,3,3,3-pentafluoro-propyl)-malonamide, (S)-2-((S)- 5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide, bis(fluoroalkyl)-1,4-benzodiazepinone, (2S)-2-hydroxy-3-methyl-N-((1S)-1-methyl-2-{[(1S)-3-methyl-2-oxo-2,3,4,5-tetrahydro-1H-3-benzazepin-1-yl]amino}-2-oxoethyl)butanamide, cis-3-[4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl] propanoic acid, dual anti-platelet study, bis(fluoroalkyl)-1,4-benzodiazepinone, or N-[1S)-2-[[(7S)-6,7-dihydro-5-methyl-6-oxo-5H-dibenz[b,d]azepin-7-yl]amino]-1-methyl-2-oxoethyl]-3,5-difluoro-benzeneacetamide.

In some embodiments, the subject is obese.

In some embodiments, the subject has hypertriglyceridemia.

In some embodiments, the hypertriglyceridemia is obesity-induced hypertriglyceridemia.

In some embodiments, the subject has fatty liver disease.

In some embodiments, the subject has non-alcoholic fatty liver disease.

In some embodiments, the subject has atherosclerosis.

In some embodiments, the subject has coronary heart disease.

In some embodiments, the subject has diabetes.

In some embodiments, the subject has Type 2 Diabetes.

In some embodiments, the subject is a human.

In some embodiments, the subject's plasma triglyceride level is >150 mg/dL.

In some embodiments, the subject's plasma triglyceride level is >500 mg/dL, about 200 to 499 mg/dL, or about 150 to 199 mg/dL.

In some embodiments, the subject's plasma triglyceride level is reduced by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, relative to the level prior to administration of the gamma-secretase inhibitor.

In some embodiments, the subject's serum triglyceride level is reduced by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, relative to the level prior to administration of the gamma-secretase inhibitor.

In some embodiments, the subject's glucose level while fasting is >100 mg/dL.

In some embodiments, the subject's glucose level two hours after eating is >140 mg/dL.

In some embodiments, the subject's plasma triglyceride level is reduced by at least 5%, at least 10%, at least 15%, at least 20%, or at least 25%, relative to the level prior to administration of the gamma-secretase inhibitor.

In some embodiments, the subject's serum triglyceride level is reduced by at least 5%, at least 10%, at least 15%, at least 20%, or at least 25%, relative to the level prior to administration of the gamma-secretase inhibitor.

Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which this invention belongs.

Terms

As used herein, “about” in the context of a numerical value or range means ±10% of the numerical value or range recited or claimed, unless the context requires a more limited range.

As used herein, “effective” when referring to an amount of a gamma-secretase inhibitor refers to the quantity of gamma-secretase inhibitor which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention.

As used herein, “treating a subject afflicted with hypertriglyceridemia” encompasses, e.g., reducing the subject's plasma or serum triglyceride level to less than 500 mg/dL, less than 200 mg/dL or less than 150 mg/dL.

As used herein, “a subject in need thereof” encompasses, e.g., a subject with plasma or serum triglyceride level greater than 150 mg/dL, greater than 200 mg/dL, or to greater than 500 mg/dL.

As used herein, a “gamma-secretase inhibitor” is an agent which reduces in vivo activity of a gamma-secretase complex. A gamma-secretase inhibitor may be, e.g., a small molecule, an anti-sense oligonucleotide, or an adenoviral vector.

Methods of Inhibiting Gamma-Secretase

In some embodiments, each compound administered to the subject is, independently, an organic compound having a molecular weight less than 1000 Daltons, a DNA aptamer, an RNA aptamer, a polypeptide, an antibody, an oligonucleotide, an interfering RNA (RNAi) molecule, a ribozyme, or a small molecule inhibitor.

In some embodiments, a compound that is capable of inhibiting gamma-secretase is administered to the subject.

In some embodiments, the compound which is capable of inhibiting gamma-secretase is an organic compound having a molecular weight less than 1000 Daltons.

Small Molecule Inhibitor

A small molecule inhibitor may administered herein to inhibit activity of gamma-secretase.

As used herein, “RO4929097” refers to 2,2-dimethyl-N-((S)-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)-N′-(2,2,3,3,3-pentafluoro-propyl)-malonamide. The CAS Registry Number for RO4929097 is 847925-91-1. The structure of RO4929097 is:

As used herein, “PF-3084014” refers to (S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H- imidazol-4-yl)pentanamide. The CAS Registry Number for PF-03084014 is 865773-15-5. The structure of PF-03084014 is:

As used herein, “BMS-708163” refers to (R)-2-(4-chloro-N-(2-fluoro-4-(1,2,4-oxadiazol-3-yl)benzyl)phenylsulfonamido)-5,5,5-trifluoropentanamide. The CAS Registry Number for BMS-708163 is 1146699-66-2. The structure of BMS-708163 is:

As used herein, “LY450139” refers to (2S)-2-hydroxy-3-methyl-N-((1S)-1-methyl-2-{[(1S)-3-methyl-2-oxo-2,3,4,5-tetrahydro-1H-3- benzazepin-1-yl]amino}-2-oxoethyl)butanamide. The CAS Registry Number for LY450139 is 425386-60-3. The structure of LY450139 is:

As used herein, “MK-0752” refers to cis-3-[4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl] propanoic acid. The CAS Registry Number for MK0752 is 471905-41-6. The structure of MK0752 is:

As used herein, dual anti-platelet study or “DAFT” refers to N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester. The CAS Registry Number for DAFT is 208255-80-5. The structure of DAFT is:

As used herein, “BMS-906024” refers to bis(fluoroalkyl)-1,4-benzodiazepinone. The CAS Registry Number for BMS-906024 is 1401066-79-2. The structure of BMS-906024 is:

As used herein, dibenzazepine or “YO-01027”, refers to N-[(1S)-2-[[(7S)-6,7-dihydro-5-methyl-6-oxo-5H-dibenz[b,d]azepin-7-yl]amino]-1-methyl-2-oxoethyl]-3,5-difluoro-benzeneacetamide. The CAS Registry Number for dibenzazepine is 209984-56-5. The structure of dibenzazepine is:

Non-limiting examples of gamma-secretase modulators which are described, for example, in the publication, Bergmans and Strooper, 2010, of which are hereby incorporated by reference in their entireties.

Non-limiting examples of gamma-secretase inhibitors which are described, for example, in the following publications: Andersson and Lendahl, 2014, Bergmans and De Strooper, 2010, Real et al., 2009, all of which are hereby incorporated by reference in their entireties.

Inhibiting Expression of Gamma-Secretase

In some embodiments, the compound which is capable of inhibiting gamma-secretase expression silences expression of a gene or silences transcription.

Oligonucleotide

Non-limiting examples of oligonucleotides capable of inhibition gamma-seretase expression include antisense oligonucleotides, ribozymes, and RNA interference molecules.

The amino acid sequence of nicastrin, NCSTN, is accessible in public databases by the GenBank accession number Q92542, and is set forth herein as SEQ ID NO: 1. The nucleotide sequence of NCSTN is also accessible in public databases by the GenBank accession number AF240468, and is set forth herein as SEQ ID NO: 2. The nucleotide sequence of NCSTN is also accessible in public databases by the GenBank accession number AK296153 and is set forth herein as SEQ ID NO: 3. The nucleotide sequence of NCSTN is also accessible in public databases by the GenBank accession number AK299142, and is set forth herein as SEQ ID NO: 4. The nucleotide sequence of NCSTN is also accessible in public databases by the GenBank accession number AK310741, and is set forth herein as SEQ ID NO: 5. The nucleotide sequence of NCSTN is also accessible in public databases by the GenBank accession number AK314764 and is set forth herein as SEQ ID NO: 6. The nucleotide sequence of NCSTN is also accessible in public databases by the accession number AY359120, and is set forth herein as SEQ ID NO: 7. The nucleotide sequence of NCSTN is also accessible in public databases by the Genbank accession number BC047621, and is set forth herein as SEQ ID NO: 8. The nucleotide sequence of NCSTN is also accessible in public databases by the GenBank accession number BC100024, and is set forth herein as SEQ ID NO: 9. The nucleotide sequence of NCSTN is also accessible in public databases by the GenBank accession number CN429672, and is set forth herein as SEQ ID NO: 10. The nucleotide sequence of nicastrin, NCSTN, is accessible in public databases by the GenBank accession number D87442, and is set forth herein as SEQ ID NO: 11.

The amino acid sequence of present in 1, PSEN1, is accessible in public databases by the GenBank accession number P49768, and is set forth herein as SEQ ID NO: 12. The amino acid sequence of PSEN1 is also accessible in public databases by the GenBank accession number AAL16811, and is set forth herein as SEQ ID NO: 13. The amino acid sequence which encodes NCSTN is accessible in public databases by the GenBank accession number CAA07825, and is set forth herein as SEQ ID NO: 14. The amino acid sequence of PSEN1 is also accessible in public databases by the GenBank accession number BAD96893, and is set forth herein as SEQ ID NO: 15. The amino acid sequence of PSEN1 is also accessible in public databases by the GenBank accession number BAH14071, and is set forth herein as SEQ ID NO: 16. The amino acid sequence which encodes PSEN1 is also accessible in public databases by the GenBank accession number BAG35430, and is set forth herein as SEQ ID NO: 17. The amino acid sequence which encodes PSEN1 is also accessible in public databases by the GenBank accession number AAH11729, and is set forth herein as SEQ ID NO: 18. The amino acid sequence which encodes PSEN1 is also accessible in public databases by the GenBank accession number AAB46416, and is set forth herein as SEQ ID NO: 19. The amino acid sequence which encodes PSEN1 is also accessible in public databases by the GenBank accession number AAB46370, and is set forth herein as SEQ ID NO: 20. The amino acid sequence which encodes PSEN1 is also accessible in public databases by the GenBank accession number AAB05894, and is set forth herein as SEQ ID NO: 21. The amino acid sequence which encodes PSEN1 is also accessible in public databases by the GenBank accession number AAB05895, and is set forth herein as SEQ ID NO: 22. The amino acid sequence which encodes PSEN1 is also accessible in public databases by the GenBank accession number CAA07825, and is set forth herein as SEQ ID NO: 23.

The amino acid sequence of presenilin 2, PSEN2, is accessible in public databases by the GenBank accession number P49810, and is set forth herein as SEQ ID NO: 24. The amino acid sequence of presenilin 2, PSEN2, is accessible in public databases by the GenBank accession number AAL16812, and is set forth herein as SEQ ID NO: 25. The amino acid sequence of PSEN2 is also accessible in public databases by the GenBank accession number BAF84988, and is set forth herein as SEQ ID NO: 26. The amino acid sequence of PSEN2 is also accessible in public databases by the GenBank accession number BAG62735, and is set forth herein as SEQ ID NO: 27. The amino acid sequence of PSEN2 is also accessible in public databases by the GenBank accession number AAH06365, and is set forth herein as SEQ ID NO: 28. The amino acid sequence of PSEN2 is also accessible in public databases by the GenBank accession number AAP35630, and is set forth herein as SEQ ID NO: 29. The amino acid sequence of PSEN2 is also accessible in public databases by the GenBank accession number AAB59557, and is set forth herein as SEQ ID NO: 30. The amino acid sequence of PSEN2 is also accessible in public databases by the GenBank accession number AAC42012, and is set forth herein as SEQ ID NO: 31. The amino acid sequence of PSEN2 is also accessible in public databases by the GenBank accession number AAC50290, and is set forth herein as SEQ ID NO: 32.

The amino acid sequence of APH1A gamma secretase subunit, APH1A, is accessible in public databases by the GenBank accession number Q96BI3, and is set forth herein as SEQ ID NO: 33. The amino acid sequence of APH1A is accessible in public databases by the GenBank accession number AAD34072, and is set forth herein as SEQ ID NO: 34. The amino acid sequence of APH1A is also accessible in public databases by the GenBank accession number AAN63816, and is set forth herein as SEQ ID NO: 35. The amino acid sequence of APH1A is also accessible in public databases by the GenBank accession number BAG51389, and is set forth herein as SEQ ID NO: 36. The amino acid sequence of APH1A is also accessible in public databases by the GenBank accession number BAC11529, and is set forth herein as SEQ ID NO: 37. The amino acid sequence of APH1A is also accessible in public databases by the GenBank accession number BAG52142, and is set forth herein as SEQ ID NO: 38. The amino acid sequence of APH1A is also accessible in public databases by the GenBank accession number BAG60040, and is set forth herein as SEQ ID NO: 39. The amino acid sequence of APH1A is also accessible in public databases by the GenBank accession number BAG60962, and is set forth herein as SEQ ID NO: 40. The amino acid sequence of APH1A is also accessible in public databases by the GenBank accession number BAG60993, and is set forth herein as SEQ ID NO: 41. The amino acid sequence of APH1A is also accessible in public databases by the GenBank accession number BAG62329, and is set forth herein as SEQ ID NO: 42. The amino acid sequence of APH1A is also accessible in public databases by the GenBank accession number CAE11677, and is set forth herein as SEQ ID NO: 43. The amino acid sequence of APH1A is also accessible in public databases by the GenBank accession number CAE11678, and is set forth herein as SEQ ID NO: 44. The amino acid sequence of APH1A is also accessible in public databases by the GenBank accession number AAM61955, and is set forth herein as SEQ ID NO: 45. The amino acid sequence of APH1A is also accessible in public databases by the GenBank accession number AAN61956, and is set forth herein as SEQ ID NO: 46. The amino acid sequence of APH1A is also accessible in public databases by the GenBank accession number AAQ89310, and is set forth herein as SEQ ID NO: 47. The amino acid sequence of APH1A is also accessible in public databases by the GenBank accession number AAH01230, and is set forth herein as SEQ ID NO: 48. The amino acid sequence of APH1A is also accessible in public databases by the GenBank accession number AAH08732, and is set forth herein as SEQ ID NO: 49. The amino acid sequence of APH1A is also accessible in public databases by the GenBank accession number AAH09501, and is set forth herein as SEQ ID NO: 50.

The amino acid sequence of APH1B gamma secretase subunit, APH1B, is accessible in public databases by the accession number Q8WW43, and is set forth herein as SEQ ID NO: 51. The amino acid sequence of APH1B is also accessible in public databases by the accession number BAD95573, and is set forth herein as SEQ ID NO: 52. The amino acid sequence of APH1B is also accessible in public databases by the accession number BAE02660, and is set forth herein as SEQ ID NO: 53. The amino acid sequence of APH1B is also accessible in public databases by the accession number AAN63817, and is set forth herein as SEQ ID NO: 54. The amino acid sequence of APH1B is also accessible in public databases by the accession number BAF83893, and is set forth herein as SEQ ID NO: 55. The amino acid sequence of APH1B is also accessible in public databases by the accession number CA366606, and is set forth herein as SEQ ID NO: 56. The amino acid sequence of APH1B is also accessible in public databases by the accession number AAQ89061, and is set forth herein as SEQ ID NO: 57. The amino acid sequence of APH1B is also accessible in public databases by the accession number AAH20905, and is set forth herein as SEQ ID NO: 58.

In some embodiments, the compound which is capable of inhibiting gamma-secretase expression is an antisense oligonucleotide, a ribozyme, or an RNA interference molecule.

Antisense Oligonucleotide

Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of target gene products in the cell.

Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5′ end of one nucleotide with the 3′ end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters.

Modifications of gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5′, or regulatory regions of the gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions −10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using “triple helix” base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (Nicholls et al., 1993, J Immunol Meth 165:81-91). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of a target polynucleotide. Antisense oligonucleotides which comprise, for example, 1, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a target polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent nucleotides, can provide sufficient, targeting specificity for a target mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides in length. Noncomplementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular target polynucleotide sequence. Antisense oligonucleotides can be modified without affecting their ability to hybridize to a target polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule. For example, internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3′, 5′-substituted oligonucleotide in which the 3′ hydroxyl group or the 5′ phosphate group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art.

Ribozymes

Ribozymes are RNA molecules with catalytic activity (Uhlmann et al., 1987, Tetrahedron. Lett. 215, 3539-3542). Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences. The coding sequence of a polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from the polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art. For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete “hybridization” region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target RNA.

Specific ribozyme cleavage sites within an RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.

Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease target gene expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or VAS element, and a transcriptional teminator signal, for controlling transcription of ribozymes in the cells (U.S. Pat. No. 5,641,673). Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.

RNA Interference

An interfering RNA RNAi molecule involves mRNA degradation. The use of RNAi has been described in Fire et al., 1998, Carthew et al., 2001, and Elbashir et al., 2001, the contents of which are incorporated herein by reference.

Interfering RNA or small inhibitory RNA (RNAi) molecules include short interfering RNAs (siRNAs), repeat-associated siRNAs (rasiRNAs), and micro-RNAs (miRNAs) in all stages of processing, including shRNAs, pri-miRNAs, and pre-miRNAs. These molecules have different origins: siRNAs are processed from double-stranded precursors (dsRNAs) with two distinct strands of base-paired RNA; siRNAs that are derived from repetitive sequences in the genome are called rasiRNAs; miRNAs are derived from a single transcript that forms base-paired hairpins. Base pairing of siRNAs and miRNAs can be perfect (i.e., fully complementary) or imperfect, including bulges in the duplex region.

Interfering RNA molecules encoded by recombinase-dependent transgenes of the invention can be based on existing shRNA, siRNA, piwi-interacting RNA (piRNA), micro RNA (miRNA), double-stranded RNA (dsRNA), antisense RNA, or any other RNA species that can be cleaved inside a cell to form interfering RNAs, with compatible modifications described herein.

As used herein, an “shRNA molecule” includes a conventional stem-loop shRNA, which forms a precursor miRNA (pre-miRNA). “shRNA” also includes micro-RNA embedded shRNAs (miRNA-based shRNAs), wherein the guide strand and the passenger strand of the miRNA duplex are incorporated into an existing (or natural) miRNA or into a modified or synthetic (designed) miRNA. When transcribed, a shRNA may form a primary miRNA (pri-miRNA) or a structure very similar to a natural pri-miRNA. The pri-miRNA is subsequently processed by Drosha and its cofactors into pre-miRNA. Therefore, the term “shRNA” includes pri-miRNA (shRNA-mir) molecules and pre-miRNA molecules.

A “stem-loop structure” refers to a nucleic acid having a secondary structure that includes a region of nucleotides which are known or predicted to form a double strand or duplex (stem portion) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion). The terms “hairpin” and “fold-back” structures are also used herein to refer to stem-loop structures. Such structures are well known in the art and the term is used consistently with its known meaning in the art. As is known in the art, the secondary structure does not require exact base-pairing. Thus, the stem can include one or more base mismatches or bulges. Alternatively, the base-pairing can be exact, i.e. not include any mismatches.

“RNAi-expressing construct” or “RNAi construct” is a generic term that includes nucleic acid preparations designed to achieve an RNA interference effect. An RNAi-expressing construct comprises an RNAi molecule that can be cleaved in vivo to form an siRNA or a mature shRNA. For example, an RNAi construct is an expression vector capable of giving rise to a siRNA or a mature shRNA in vivo. Non-limiting examples of vectors that may be used in accordance with the present invention are described herein and will be well known to a person having ordinary skill in the art. Exemplary methods of making and delivering long or short RNAi constructs can be found, for example, in WO001/68836 and WO01/75164.

RNAi is a powerful tool for in vitro and in vivo studies of gene function in mammalian cells and for therapy in both human and veterinary contexts. Inhibition of a target gene is sequence-specific in that gene sequences corresponding to a portion of the RNAi sequence, and the target gene itself, are specifically targeted for genetic inhibition. Multiple mechanisms of utilizing RNAi in mammalian cells have been described. The first is cytoplasmic delivery of siRNA molecules, which are either chemically synthesized or generated by DICER-digestion of dsRNA. These siRNAs are introduced into cells using standard transfection methods. The siRNAs enter the RISC to silence target mRNA expression.

Another mechanism is nuclear delivery, via viral vectors, of gene expression cassettes expressing a short hairpin RNA (shRNA). The shRNA is modeled on micro interfering RNA (miRNA), an endogenous trigger of the RNAi pathway (Lu et al., 2005, Advances in Genetics 54: 117-142, Fewell et al., 2006, Drug Discovery Today 11: 975-982). Conventional shRNAs, which mimic pre-miRNA, are transcribed by RNA Polymerase II or III as single-stranded molecules that form stem-loop structures. Once produced, they exit the nucleus, are cleaved by DICER, and enter the RISC as siRNAs.

Another mechanism is identical to the second mechanism, except that the shRNA is modeled on primary miRNA (shRNAmir), rather than pre-miRNA transcripts (Fewell et al., 2006). An example is the miR-30 miRNA construct. The use of this transcript produces a more physiological shRNA that reduces toxic effects.

The shRNAmir is first cleaved to produce shRNA, and then cleaved again by DICER to produce siRNA. The siRNA is then incorporated into the RISC for target mRNA degradation. However, aspects of the present invention relate to RNAi molecules that do not require DICER cleavage. See, e.g., U.S. Pat. No. 8,273,871, the entire contents of which are incorporated herein by reference.

For mRNA degradation, translational repression, or deadenylation, mature miRNAs or siRNAs are loaded into the RNA Induced Silencing Complex (RISC) by the RISC-loading complex (RLC). Subsequently, the guide strand leads the RISC to cognate target mRNAs in a sequence-specific manner and the Slicer component of RISC hydrolyses the phosphodiester bound coupling the target mRNA nucleotides paired to nucleotide 10 and 11 of the RNA guide strand. Slicer forms together with distinct classes of small RNAs the RNAi effector complex, which is the core of RISC. Therefore, the “guide strand” is that portion of the double-stranded RNA that associates with RISC, as opposed to the “passenger strand,” which is not associated with RISC.

It is not necessary that there be perfect correspondence of the sequences, but the correspondence must be sufficient to enable the RNA to direct RNAi inhibition by cleavage or blocking expression of the target mRNA. In preferred RNA molecules, the number of nucleotides which is complementary to a target sequence is 16 to 29, 18 to 23, or 21-23, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.

Isolated RNA molecules can mediate RNAi. That is, the isolated RNA molecules of the present invention mediate degradation or block expression of mRNA that is the transcriptional product of the gene. For convenience, such mRNA may also be referred to herein as mRNA to be degraded. The terms RNA, RNA molecule(s), RNA segment(s) and RNA fragment(s) may be used interchangeably to refer to RNA that mediates RNA interference. These terms include double-stranded RNA, small interfering RNA (siRNA), hairpin RNA, single-stranded RNA, isolated RNA (partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA), as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA). Nucleotides in the RNA molecules of the present invention can also comprise nonstandard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides. Collectively, all such altered RNAi molecules are referred to as analogs or analogs of naturally-occurring RNA. RNA of the present invention need only be-sufficiently similar to natural RNA that it has the ability to mediate RNAi.

As used herein the phrase “mediate RNAi” refers to and indicates the ability to distinguish which mRNA molecules are to be afflicted with the RNAi machinery or process. RNA that mediates RNAi interacts with the RNAi machinery such that it directs the machinery to degrade particular mRNAs or to otherwise reduce the expression of the target protein. In one embodiment, the present invention relates to RNA molecules that direct cleavage of specific mRNA to which their sequence corresponds. It is not necessary that there be perfect correspondence of the sequences, but the correspondence must be sufficient to enable the RNA to direct RNAi inhibition by cleavage or blocking expression of the target mRNA.

In some embodiments, an RNAi molecule of the invention is introduced into a mammalian cell in an amount sufficient to attenuate target gene expression in a sequence specific manner. The RNAi molecules of the invention can be introduced into the cell directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to the cell. In certain embodiments the RNAi molecule can be a synthetic RNAi molecule, including RNAi molecules incorporating modified nucleotides, such as those with chemical modifications to the 2′-OH group in the ribose sugar backbone, such as 2′-O-methyl (2′OMe), 2′-fluoro (2′ F) substitutions, and those containing 2′OMe, or 2′F, or 2′-deoxy, or “locked nucleic acid” (LNA) modifications. In some embodiments, an RNAi molecule of the invention contains modified nucleotides that increase the stability or half-life of the RNAi molecule in vivo and/or in vitro. Alternatively, the RNAi molecule can comprise one or more aptamers, which interact(s) with a target of interest to form an aptamer:target complex. The aptamer can be at the 5′ or the 3′ end of the RNAi molecule. Aptamers can be developed through the SELEX screening process and chemically synthesized. An aptamer is generally chosen to preferentially bind to a target. Suitable targets include small organic molecules, polynucleotides, polypeptides, and proteins. Proteins can be cell surface proteins, extracellular proteins, membrane proteins, or serum proteins, such as albumin. Such target molecules may be internalized by a cell, thus effecting cellular uptake of the shRNA. Other potential targets include organelles, viruses, and cells.

As noted above, the RNA molecules of the present invention in general comprise an RNA portion and some additional portion, for example a deoxyribonucleotide portion. The total number of nucleotides in the RNA molecule is suitably less than in order to be effective mediators of RNAi. In preferred RNA molecules, the number of nucleotides is 16 to 29, more preferably 18 to 23, and most preferably 21-23.

Adenoviral Vector

An adenoviral vecor encodes an oligonucleotide. The use of adenoviral vectors in gene therapy and tissue-specific targeting has been described in Beatty and Curiel, 2012, Barnett et al., 2002, and Rots et al., 2003, the contents of which are incorporated herein by reference.

Methods of Administration

“Administering” compounds in embodiments of the invention can be effected or performed using any of the various methods and delivery systems known to those skilled in the art. The administering can be, for example, intravenous, oral; intramuscular, intravascular, intra-arterial, intracoronary, intramyocardial, intraperitoneal, and subcutaneous. Other non-limiting examples include topical administration, or coating of a device to be placed within the subject.

Injectable Drug Delivery

Injectable drug delivery systems may be employed in the methods described herein include solutions, suspensions, gels.

Oral Drug Delivery

Oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc). Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents (e.g., gums, zanthans, cellulosics and sugars), humectants (e.g.; sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid), anti-caking agents, coating agents, and chelating agents (e.g., EDTA).

For oral administration in liquid dosage form, a gamma-secretase inhibitor may be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like.

Naked Administration

The compounds used in embodiments of the present invention can be administered by naked administration.

Pharmaceutically Acceptable Carrier

The compounds used in embodiments of the present invention can be administered in a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier” is a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the compounds to the subject. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Liposomes such as small unilamellar vesicles, large unilamallar vesicles, and multilamellar vesicles are also a pharmaceutically acceptable carrier. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines. The compounds may be administered as components of tissue-targeted emulsions. Examples of lipid carriers for antisense delivery are disclosed in U.S. Pat. Nos. 5,855,911 and 5,417,978, which are incorporated herein by reference. The compounds used in the methods of the present invention can be administered in admixture with suitable pharmaceutica diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral, rectal, topical, intravenous or direct injection or parenteral administration. The compounds can be administered alone or mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. The active agent can be co-administered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

A compound of the invention can be administered in a mixture with suitable pharmaceutical diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral, rectal, topical, intravenous or direct injection or parenteral administration. The compounds can be administered alone but are generally mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. In one embodiment the carrier can be a monoclonal antibody. The active agent can be co-administered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

Specific examples of pharmaceutical acceptable carriers and excipients that may be used to formulate oral dosage forms of the present invention are described in U.S. Pat. No. 3,903,297, issued Sep. 2, 1975.

Tablets

Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. For instance, for oral administration in the dosage unit form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

Specific Administration To Liver

Embodiments of the invention relate to specific administration to the liver or hepatocytes.

In some embodiments, a compound may specifically target the liver.

In some embodiments, a compound may specifically target hepatocytes.

In some embodiments, a compound may be specifically targeted to the liver by coupling the compound to ligand molecules, targeting the compound to a receptor on a hepatic cell, or administering the compound by a bio-nanocapsule.

A compound of the invention can also be administered by coupling of ligand molecules, such as coupling or targeting moieties on preformed nanocarriers, such as (PGA-PLA nanoparticles, PLGA nanoparticles, cyclic RGD-doxorubicin-nanoparticles, and poly(ethylene glycol)-coated biodegradable nanoparticles), by the post-insertion method, by the Avidin-Biotin complex, or before nanocarriers formulation, or by targeting receptors present on various hepatic cell, such as Asialoglycoproein receptor (ASGP-R), HDL-R, LDL-R, IgA-R, Scavenger R, Transferrin R, and Insulin R, as described in: Mishra et al., (2013) Efficient Hepatic Delivery of Drugs: Novel Strategies and Their Significance, BioMed Research International 2013: 382184, dx.doi.org/10.1155/2013/382184, the entire contents of which are incorporated herein by reference.

A compound of the invention can also be administered by bio-nanocapsule, as described in: Yu et al., (2005) The Specific delivery of proteins to human liver cells by engineered bio-nanocapsules, FEBS Journal 272: 3651-3660, dx.doi.org/10.1111/j.1742-4658.2005.04790.x, the entire contents of which are incorporated herein by reference.

In some embodiments, an oligonucleotide specifically targets the liver.

In some embodiments, an oligonucleotide specifically targets hepatocytes.

Antisense oligonucleotides of the invention can also be targeted to hepatocytes, as described in: Prakash et al., (2014) Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice, Nucleic Acids Research 42(13): 8796-8807, dx.doi.org/10,1093/nar/gku531, the entire contents of which are incorporated herein by reference.

As used herein, the term “effective amount” refers to the quantity of a component that is sufficient to treat a subject without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention, i.e. a therapeutically effective amount. The specific effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of subject being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.

Techniques and compositions for making dosage forms useful in the present invention are described in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol. 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.

The dosage of a compound of the invention administered in treatment will vary depending upon factors such as the pharmacodynamic characteristics of the compound and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with; and the desired therapeutic effect.

A dosage unit of the compounds of the invention may comprise a compound alone, or mixtures of a compound with additional compounds used to treat cancer. The compounds can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by injection or other methods, into the eye, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.

In an embodiment, the gamma-secretase inhibitor may be administered once a day, twice a day, every other day, once weekly, or twice weekly.

In an embodiment, 0.01 to 1000 mg of a gamma-secretase inhibitor is administered per administration.

A subject's triglyceride level may be expressed herein as plasma triglyceride or serum triglyceride.

A subject's apolipoprotein C3 (ApoC3) level may be expressed herein as plasma ApoC3 or serum ApoC3.

Where a range is given in the specification it is understood that the range includes all integers and 0.1 units within that range, and any sub-range thereof. For example, a range of 1 to 5 is a disclosure of 1.0, 1.1, 1.2, etc.

This invention will be better understood by reference to the Examples which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.

Example 1. Pharmacologic Notch Inhibition Decreases Blood Glucose in Lean and Obese Mice Without Affecting Weight

The Notch signaling pathway is a highly conserved cell signaling system present in most multicellular organisms. Notch signaling is well-established as critical for cell-cell communication and control of differentiation during normal development. Notch signaling is frequently upregulated in tumors, and a variety of Notch inhibitors are in clinical development, some as advanced as Phase I/II trials, for cancer. Notch stimulates mTorc1 activity in T-cell leukemia, and reduction in Notch signaling with gamma-secretase inhibitor (GSI) treatment blocks mTorc1 activation, and tumorigenesis, as illustrated in FIG. 1. Gamma-secretase inhibitors (GSIs) are pharmacologic inhibitors of Notch signaling (FIG. 2).

There are interactions between Notch signaling and key metabolic mediators of obesity-related disease, and hepatic Notch signaling is elevated in mouse models and patients with Type 2 Diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). We hypothesized that Notch may reciprocally affect FoxO1- or mTorc1-dependent signaling, and thus carbon flux towards glucose and lipid production, and hypothesized that Notch inhibitors may be repurposed for treatment of diabetes and fatty liver disease.

Lean mice, diet induced obese (DIO) mice, and leptin-deficient obese (ob/ob) mice were administered either vehicle or GSI. GSI treatment decreased blood glucose in lean and obese mice without affecting weight as shown in Table 1.

TABLE 1 Lean mice, diet induced obese (DIO) mice, and leptin- deficient obese (ob/ob) mice administered gamma secretase inhibitor (GSI), dibenzazepine (DBZ). Vehicle control for all experiments is normal saline containing 0.5% methoxycellulose/0.1% Tween-80. Weight Glucose Insulin Cohort Treatment (grams) (mg/dl) (ng/ml) lean vehicle 22 ± 0.5  79 ± 4  0.4 ± 0.15 GSI 22 ± 0.6  64 ± 2 *** 0.49 ± 0.04 DIO vehicle 34 ± 0.9 135 ± 12 1.62 ± 1.18 GSI 33 ± 0.7  72 ± 5 ***  1.3 ± 0.32 ob/ob vehicle 49 ± 0.9 313 ± 33 23.2 ± 3.11 GSI 48 ± 0.9  98 ± 6 *** 15.6 ± 3.25 * * p < 0.05 ** p < 0.01 *** p < 0.001 vs. vehicle

GSI (which will be used interchangeably with the specific drug name, dibenzazepine, DBZ) treatment reduced both both fasted as well as refed (or exogenous) glucose in DIO mice as compared to vehicle (normal saline containing 0.5% methoxycellulose/0.1% Tween-80) (FIG. 3). Treatment of mice with the gamma-secretase inhibitor, dibenzazepine (DBZ) reduces fasted or refed plasma glucose (FIG. 3 Panel A), and improves glucose clearance (FIG. 3 Panel B).

Example 2. GSI Lowers Serum Triglycerides (TG)

Mice were administered GSI or vehicle alone to determine the effect of GSI treatment on serum triglyceride levels.

Mice administered GSI had lower plasma triglycerides than mice administered vehicle alone (FIG. 4). DBZ-treated obese mice, diet-induced obese (DIO) and leptin-deficient obese (ob/ob), show lower plasma triglyceride (TG) as compared to vehicle treatment. Analysis of VLDL, LDL, and HDL fractions showed that GSI lowered triglycerides in the VLDL fraction (FIG. 5). Lower plasma TG levels with low dose DNZ was not associated with apparent gastrointestinal (GI) toxicity (FIG. 6).

DBZ-induced lower plasma TG in diet-induced obese (DIO) and leptin-deficient obese (ob/ob) mice is in the VLDL fraction. GSI treated mice showed normal triglyceride secretion (FIG. 7). Normal plasma TG levels after lipoprotein lipase inhibition with Poloxamer 407 (P407) indicates that DBZ does not affect TG secretion. GSI treated mice showed less plasma triglyceride excursion after lipid gavage compared to mice administered vehicle only (FIG. 8). Lower plasma TG levels after oral olive oil gavage indicates that DBZ increases TG clearance. The combination of these pieces of data (normal TG secretion, less fasted TG and lower TG in serum after gavage) suggests either: (1) Adipose phenotype, i.e., less lipolysis of fat stores, or (2) Liver phenotype, i.e., increased TG uptake from circulation. To differentiate these—we created a liver-specific gamma-secretase knockout mouse.

Example 3. Hepatocyte-Specific Gamma-Secretase Deficiency Reduces Plasma Triglycerides

To elucidate the mechanism of the results of Example 2, a mouse that had gamma-secretase deficiency specifically in hepatocytes was created (Albumin-cre:Nicastrin fl/fl mice, henceforth L-Ncst).

L-Ncst mice showed similar glucose improvement to GSI-treated mice according to glucose tolerance test (GTT) and pyruvate tolerance test (PTT) (FIG. 9). L-Ncst (hepatocyte-specific gamma-secretase knockout) mice showed improved glucose clearance as compared to Cre- control mice, similar GSI-treatment, when challenged with either an intraperitoneal glucose (GTT) or pyruvate (PTT) load.

Chow-fed L-Ncst mice had lower plasma TG (FIG. 10). These data prove that liver γ-secretase is involved in TG clearance from circulation, or in the production of a secreted protein (hepatokine) that alters TG metabolism. Of these, the likeliest target is ApoC3, an apolipoprotein produced exclusively in liver that has been proven to affect TGs. People or mice with ApoC3 deficiency show very low plasma TG, and low risk for coronary disease (CAD). Conversely, excessive production of ApoC3 is associated with high serum TG and excess CAD.

High fat diet (HFD) fed L-Ncst mice had lower refed serum triglycerides compared to control mice (FIG. 11). L-Ncst mice showed lower serum TG compared to Cre- control mice, similar to GSI-treated mice. HFD feeding increased the difference between Cre- and L-Ncst mice in serum TG. Lower p triglycerides were observed in the VLDL fraction (FIG. 12). As with GSI treatment, reduced plasma TG seen in L-Ncst mice as compared to Cre- control mice is in the VLDL fraction. Both fasted, but more markedly refed plasma triglycerides, were lower in L-Ncst mice compared to control mice (FIG. 13). Both fasted and refed serum TG are lower in L-Ncst mice than in Cre- control mice. Triglyceride secretion was unchanged in HFD-fed L-Ncst mice compared to control mice (FIG. 14). As with GSI treatment, comparable serum TG levels after lipoprotein lipase inhibition with Poloxamer 407 (P407) indicates that L-Ncst mice show similar TG secretion as Cre- control mice. As with GSI treatment, serum TG levels after olive oil gavage in L-Ncst mice as compared to Cre- controls proves that L-Ncst mice show increased TG clearance (FIG. 15).

Example 4. Lower Serum TG Observed in L-Ncst Mice is by Lower ApoC3

A gene expression analysis of hepatic genes that affect serum triglycerides showed that only Apoc3 expression was altered in the L-Ncst mice (FIG. 16). Serum ApoC3 levels were lower in HFD-fed L-Ncst mice compared to Cre- control mice (FIG. 17). Serum levels of the apolipoprotein ApoC3 levels were lower in both HDL and VLDL fractions in HFD-fed L-Ncst mice compared to Cre- control mice (FIG. 18). Hepatic ApoC3 levels were also decreased in L-Ncst mice compared to control mice (FIG. 19).

In fasted and refed L-Ncst mice, ApoC3 protein expression was was decreased in serum and liver compared to fasted and refed control mice (FIG. 20). Serum levels of the apolipoprotein ApoC3 were lower in L-Ncst mice even though liver mRNA and protein for ApoC3 were unaffected. Also, Psen2 protein expression was was decreased in liver, but no change in hepatic protein, ApoB100/48 in fasted and refed L-Ncst mice compared to control mice. Correspondingly, there was no change in Apoc3 mRNA in chow-fed L-Ncst mice (FIG. 21).

Serum ApoC3 correlated with plasma TG, but hepatic ApoC3 and Apoc3 mRNA did not correlate with plasma TG (FIG. 22). These data suggest that liver gamma-secretase is involved in either ApoC3 secretion or clearance from circulation, as only serum ApoC3 (but not hepatic Apoc3 mRNA or hepatic ApoC3 protein) are reduced in L-Ncst mice.

Example 5. Adenoviral Transduction of Cre- Control and L-Ncst Mice and shRNA Mediated Knockdown in Rat Hepatocytes

Liver ApoC3 knockdown (with adeno-delivered shRNA) eliminates difference between HFD-fed Cre- and L-Ncst mice (FIG. 23). Serum, but not hepatic, ApoC3 protein levels correlate with plasma TG. These data prove that lower serum TG observed in L-Ncst mice is by lower ApoC3. Future work will be to determine the mechanism by which this happens.

Adenoviral transduction of L-Ncst mice with ApoC3 increases serum TG to levels comparable to Cre- control mice (FIG. 24). shRNA-mediated knockdown of Nicastrin (sequence of shRNA: CTCCTTCCACAATCGGTATTA) in rat hepatocytes reduces ApoC3 secretion (FIG. 25).

Discussion

Hypertriglyceridemia is not easily treated. Currently available therapies include fibrates (fenofibrate, gemfibrozil) and other less potent triglyceride-lowering agents such as bile acid sequesterants, niacin and statins. Fibrates have been shown to reduce cardiovascular risk, but many patients are unable to reach plasma triglyceride treatment goals with these medications. As such, novel molecular targets to reduce plasma triglycerides have been long-sought.

Recent work has shown that the liver-secreted apolipoprotein, ApoC3, may impact plasma triglyceride levels. Humans with genetic variants that confer partial ApoC3 deficiency, including several Amish and Ashkenazi Jewish populations, exhibit lower plasma triglyceride levels, leading to lower risk of coronary heart disease (Pollin T I, Science, 2008). These human studies have been confirmed with mouse data—ApoC3 knockout mice demonstrate markedly lower plasma triglyceride levels (Jong et al, J Lipid Res, 2001), whereas mouse models of ApoC3 overexpression given rise to massive hypertriglyceridemia and excess atherosclerosis (Masucci-Magoulas L, et al, Science, 1997). This data was so compelling that various pharmaceutical companies are targeting ApoC3 as a potentially novel means to reduce plasma triglycerides (Gaudet D et al., N Engl J Med, 2014), and hopefully reduce atherosclerosis and consequent coronary heart disease.

The above Examples show that inhibition of the gamma-secretase complex in liver reduces both liver and circulating ApoC3, leading to lower plasma triglycerides. Gamma-secretase is a enzymatic complex composed of targeting (Nicastrin), catalytic (Presenlin) as well as regulatory subunits (Aph1, PEN2) (Tolia and De Strooper, Semin Cell Dev Biol, 2009). This enzyme is the prototype for intramembrane proteases, and its known targets include Notch receptors, Alzheimer's precursor protein (APP), and others (De Strooper and Annaert, Annu Rev Cel Dev Biol, 2010). Gamma-secretase inhibitors were developed, in part, to reduce APP cleavage to beta-amyloid, in a failed attempt to treat Alzheimer's disease (De Strooper B et al., Nat Rev Neurol, 2010). The

(Andersson and Lendahl, Nat Rev Drug Disc, 2014), and perhaps even for Alzheimer's Disease (Imbimbo B P et al., Expert Opin Investig Drugs, 2011), but unlikely for chronic treatment of hypertriglyceridemia. As such, liver-specific inhibitors of the gamma-secretase, by methods described above, would be advantageous to maintain efficacy while limiting or even eliminating GI toxicity.

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What is claimed is:
 1. A method of reducing a subject's plasma triglyceride level, comprising administering to a subject in need thereof a gamma-secretase inhibitor in an amount effective to reduce the subject's plasma triglyceride level.
 2. A method of treating a subject afflicted with hypertriglyceridemia, comprising administering to the subject a gamma-secretase inhibitor in an amount effective to treat the subject.
 3. The method of claim 1 or claim 2, wherein the administration reduces the subject's serum triglyceride level.
 4. The method of claim 3, wherein the administration reduces the triglyceride level in the subject's very low-density lipoprotein (VLDL) serum fraction.
 5. The method of claim 3, wherein the administration reduces the subject's serum triglyceride level and serum apolipoprotein C3 (ApoC3) level.
 6. A method of reducing a subject's plasma glucose level, comprising administering to a subject in need thereof a gamma-secretase inhibitor in an amount effective to reduce the subject's glucose level.
 7. The method of any one of claims 1-6, wherein administration of the gamma-secretase inhibitor inhibits whole-body gamma-secretase.
 8. The method of any one of claims 1-6, wherein administration of the gamma-secretase inhibitor inhibits liver gamma-secretase without significantly inhibiting gamma-secretase elsewhere in the subject.
 9. The method of any one of claims 1-6, wherein the administration of the gamma-secretase inhibitor targets the gamma-secretase inhibitor to the liver.
 10. The method of claim 10, wherein the administration of the gamma-secretase inhibitor targets the gamma-secretase inhibitor to hepatocytes.
 11. The method of any one of claims 8-10, wherein the gamma-secretase inhibitor is (i) coupled to a ligand molecule targeted to a receptor on a hepatic cell, or (ii) administered by a bio-nanocapsule.
 12. The method of any one of claims 8-11, wherein gastrointestinal Notch inhibition is substantially uninhibited.
 13. The method of any one of claims 1-12, wherein the gamma-secretase inhibitor is a small molecule inhibitor, an oligonucleotide or an adenoviral vector.
 14. The method of claim 13, wherein the gamma-secretase inhibitor is an oligonucleotide.
 15. The method of claim 14, wherein the oligonucleotide is an antisense oligonucleotide, an RNA-interference inducing compound, or a ribozyme.
 16. The method of claim 14 or claim 15, wherein the oligonucleotide is targeted to hepatocytes.
 17. The method of any one of claims 14-16, wherein the oligonucleotide comprises 1, 2, 3, 4, or 5 or more stretches of nucleotides in a sequence that is complementary to nicastrin-encoding mRNA, presenilin 1-encoding mRNA and presenilin 2-encoding mRNA, or APH1A-encoding mRNA and API1B-encoding mRNA, wherein each stretch of complementary continguous nucleotides is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides in length.
 18. The method of any one of claims 14-17, wherein the oligonucleotide is modified to increase its stability in vivo.
 19. The method of claim 13, wherein the gamma-secretase inhibitor is a small molecule inhibitor.
 20. The method of claim 19, wherein the small molecule inhibitor is 2,2-dimethyl-N-((S)-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)-N′-(2,2,3,3,3-pentafluoro-propyl)-malonamide, (S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide, bis(fluoroalkyl)-1,4- benzodiazepinone, (2S)-2-hydroxy-3-methyl-N-((1S)-1-methyl-2-{[(1S)-3-methyl-2-oxo-2,3,4,5-tetrahydro-1H-3-benzazepin-1-yl]amino}-2- oxoethyl)butanamide, cis-3-[4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl] propanoic acid, dual anti-platelet study, bis(fluoroalkyl)-1,4-benzodiazepinone, or N-[(1S)-2-[[(7S)-6,7-dihydro-5-methyl-6-oxo-5H-dibenz[b, d]azepin-7-yl]amino]-1-methyl-2-oxoethyl]-3,5-difluoro-benzeneacetamide.
 21. The method of any one of claims 1-20, wherein the subject is obese.
 22. The method of any one of claims 1-21, wherein the subject has hypertriglyceridemia.
 23. The method of claim 22, wherein the hypertriglyceridemia is obesity-induced hypertriglyceridemia.
 24. The method of any one of claims 1-23, wherein the subject has fatty liver disease.
 25. The method of claims 24, wherein the subject has non-alcoholic fatty liver disease.
 26. The method of any one of claims 1-25, wherein the subject has atherosclerosis.
 27. The method of any one of claims 1-26, wherein the subject has coronary heart disease.
 28. The method of any one of claims 1-27, wherein the subject has diabetes.
 29. The method of claim 28, wherein the subject has Type 2 Diabetes.
 30. The method of any one of claims 1-29, wherein the subject is a human.
 31. The method of any one of claims 1-30, wherein the subject's plasma triglyceride level is >150 mg/dL.
 32. The method of any one of claims 1-30, wherein the subject's plasma triglyceride level is >500 mg/dL, about 200 to 499 mg/dL, or about 150 to 199 mg/dL.
 33. The method of any one of claims 1-32, wherein the subject's plasma triglyceride level is reduced by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, relative to the level prior to administration of the gamma-secretase inhibior.
 34. The method of any one of claims 1-33, wherein the subject's glucose level while fasting is >100 mg/dl,.
 35. The method of any one of claims 1-33, wherein the subject's glucose level two hours after eating is >140 mg/dL.
 36. The method of any one of claims 1-35, wherein the subject's plasma triglyceride level is reduced by at least 5%, at least 10%, at least 15%, at least 20%, or at least 25%, relative to the level prior to administration of the gamma-secretase inhibitor. 